胰岛素样生长因子-1对氧化损伤的Schwann细胞抗凋亡的实验研究
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摘要
目的:施万细胞(Schwann cell,SCs)是周围神经的主要结构和功能细胞,是周围神经系统特有的一种胶质细胞,在神经损伤后的再生和功能恢复中起着重要的作用。长期以来,周围神经病变严重影响着人们的生活质量,是神经科学研究领域中的一个复杂但十分重要的病变过程。近年来的研究发现,其发病机理之一是氧化应激引起氧自由基增多,从而导致了Schwann细胞的凋亡。Schwann细胞能够包绕轴突形成有髓纤维或无髓纤维,并分泌多种神经因子参与神经系统的发育和损伤后的再生。因此,越来越多的研究通过施加外界因素(如各种神经生长因子、中药、电磁场、硅胶管及其他移植物等)干预Schwann细胞的生存环境,探讨能够促进周围神经损伤后的再生过程的适宜方法。
目前,应用神经营养因子促进神经系统生长发育的研究已经得到人们广泛的认可。胰岛素样生长因子—1(insulin-like growth factor-1,IGF-1)是一种肽能神经生长因子,与组织分化、增殖和成熟密切相关。IGF-1具有非选择性神经营养作用,能促进外周神经再生。体外研究发现,IGF-1对各种中枢神经细胞均有神经营养和保护作用,能够影响神经细胞的生长、存活和分化。同时,大量实验证实,IGF-1对多种刺激因素(如缺血、缺氧,氧化应激等)引起的细胞凋亡有抑制作用。IGF-1的抗氧化作用已有初步研究,但对周围神经中Schwann细胞氧化损伤的保护作用的机理尚未见报道。
本课题通过建立过氧化氢诱导的Schwann细胞氧化损伤模型,并在此模型的基础上,观察了神经因子IGF-1对氧化损伤的Schwann细胞的保护作用,通过MTT、生化检测、吖叮橙染色、Western blot等方法进行细胞活力测定、酶活力分析、凋亡率的分析以及凋亡蛋白Bcl-2表达水平的测定,探讨IGF-1抗氧化损伤、抗凋亡的作用机理,为揭示IGF-1治疗周围神经病变的机制提供理论依据。
方法:本课题从新生一周内Wistar大鼠的坐骨神经和臂丛神经获取Schwann细胞,用含10%胎牛血清(FCS)的DMEM/F12培养液于5%CO_2、37℃、饱和湿度的细胞培养箱中培养。Schwann细胞培养72小时后,分为3个实验组,另设一空白对照组。
1.大体观察在加入H_2O_2损伤因素与IGF-1保护因素后12h时,倒置相差显微镜下观察Schwann细胞的大体形态、贴壁数量和生长状态等情况,并照相记录。
2.Schwann细胞的纯化及鉴定利用传代法纯化Schwann细胞。实验前Schwann细胞爬片作S-100免疫组化染色,鉴定Schwann细胞。
3.实验分组将培养细胞分为3组:①过氧化氢损伤组:在培养基中加入终浓度为100μmol/L的H_2O_2;②IGF-1保护组:100μmol/L的H_2O_2+100ng/ml的IGF-1;③正常组(不加任何因素);同时设立仅加培养液的空白对照组。
4.MTT法进行胰岛素样生长因子-1(IGF-1)抗过氧化氢氧化损伤的作用分析。
5.生化检测细胞内脂质过氧化物MDA和超氧化物歧化酶SOD的水平。
6.应用吖叮橙荧光染色观察、比较过氧化氢损伤组、IGF-1保护组和正常对照组细胞的存活、凋亡情况。
7.正常对照组、过氧化氢损伤组和IGF-1保护组细胞提取总蛋白,通过SDS-PAGE及蛋白印迹(Western blot)检测凋亡蛋白Bcl-2的表达水平变化情况。
通过以上的实验,建立了过氧化氢诱导Schwann细胞的氧化损伤模型,在此模型的基础上探讨IGF-1对Schwann细胞抗氧化损伤、抗凋亡的作用机理。
结果:刚接种的原代Schwann细胞呈圆形,悬浮在培养液中,接种6h后大部分细胞贴壁,部分细胞开始呈椭圆形,24h后变成双极,胞体饱满,立体感强,少数呈三角形,伸出三个突起。培养48～72h后,出现聚合现象,许多细胞聚合在一起,呈端对端、肩并肩、漩涡状或栅栏状排列,在细胞密度较低时,则聚集成堆,形成“细胞岛”,免疫细胞化学染色为S-100阳性;另一种细胞稍大,外形不规则,呈扁平状,突起短而多,核较浅,为成纤维细胞,所占比例较低。培养72小时后进行传代、纯化Schwann细胞,镜下观察Schwann细胞的纯度明显增加,免疫化学染色显示80%以上的细胞S-100呈阳性反应。加入H_2O_2 12小时后,在倒置显微镜下观察发现细胞的形态发生改变,细胞突起萎缩,胞体肿胀变圆、空泡化变,细胞数量减少,部分细胞脱壁,漂浮于培养液中,胞体立体感差,折光度下降,并可见大量细胞碎片;IGF-1保护组的细胞生长状态良好,突起伸展,细胞存活数量多,胞体较饱满、立体感较强,少见细胞皱缩破碎现象,但细胞聚团现象较明显;正常组的细胞胞体梭形、折光度好,突起较长,细胞数量明显较多,偶有漂浮死细胞。
不同实验组检测结果如下:
1.MTT法检测IGF-1抗氧化损伤的作用分析表明:IGF-1可明显拮抗过氧化氢诱导的氧化损伤,与过氧化氢损伤组相比,IGF-1保护组可维持较高的细胞存活率,二者之间有统计学意义(P＜0.05)。同时,IGF-1保护组的细胞存活率低于正常组,二者之间亦有统计学意义。
2.生化指标SOD检测结果表明:与过氧化氢损伤组相比,IGF-1保护组细胞内超氧化物歧化酶(SOD)维持在较高的活力水平,表明IGF-1可明显拮抗过氧化氢引起的SOD降低;同时低于正常对照组,有统计学意义(P＜0.05)。
3.生化指标MDA检测结果表明:与正常组相比,过氧化氢引起细胞内脂质过氧化物的大量堆积,即MDA含量增加;而IGF-1可明显降低细胞内脂质过氧化物的含量(P＜0.05)。
4.吖叮橙荧光染色法在荧光显微镜下观察到,发生凋亡的阳性细胞呈圆形,体积明显缩小,细胞核内可见致密浓染的黄色荧光和凋亡小体,胞质为火焰样橘红色荧光;正常组的Schwann细胞呈梭形,胞核圆形、椭圆形,呈亮绿色,胞质为灰绿色和橙红色,亦可见少量的凋亡前期细胞。损伤组细胞量明显减少,凋亡形态典型;保护组存活细胞量增多,细胞状态明显好转。
5.蛋白印迹Western blot显示抗凋亡蛋白Bcl-2的表达情况:过氧化氢损伤组细胞中抗凋亡蛋白Bcl-2的表达明显下调,而加入IGF-1保护后Bcl-2蛋白表达上调(P＜0.05),正常组的蛋白表达量最高。
结论:我们以原代培养的Schwann细胞为实验材料,利用过氧化氢诱导Schwann细胞建立氧化损伤模型,在此基础上,观察过氧化氢诱导Schwann细胞的氧化损伤作用及神经因子IGF-1对氧化损伤的Schwann细胞的保护作用。实验结果显示:过氧化氢损伤能够引起细胞内自由基的增多,SOD活力的降低,脂质过氧化物的增多,并且自由基的增多能通过信号通路上抑制凋亡蛋白Bcl-2的表达而最终引起细胞的凋亡;IGF-1不仅能降低细胞内脂质过氧化物(MDA)的含量,维持细胞内超氧化物歧化酶(SOD)的含量,还能通过上调抗凋亡蛋白Bcl-2的表达来抑制细胞死亡的发生,证明IGF-1可以明显拮抗过氧化氢对Schwann细胞的氧化损伤作用。
本研究探讨了IGF-1对Schwann细胞氧化损伤的保护作用,从氧化损伤信号转导通路方面探讨了IGF-1的抗氧化、抗凋亡作用的分子机制,为研究周围神经病变中自由基的分子作用机制以及IGF-1等神经营养因子在在周围神经病变中的治疗作用提供了一定的理论依据。
Purpose:Schwann cell is a major glial cell of peripheral nerve system on architecture and function.It plays an important part in the regeneration and functional restoration after the nerve injured.For many years,the pathological changes of peripheral nerve affect the people's living quality badly,which is a very important and complicate process in neural system diseases.Recently,researchers found that one of the pathogenesis is that oxidative irritation can make free radical increase,which cause the apoptosis of Schwann cells.Schwann cell can wrap the axon to form myelinated nerve fiber or nonmyelinated nerve fiber.It can secrete many kinds of neurotrophic factors which are concerned with development and regeneration of nerve.Recently, more and more people apply external assists:such as neurotrophic factor,Chinese traditional medicine,electromagnetism,silica gel and other grafts to enhance chwann cells' function,then to cure the peripheral nerve injuries.
Nowadays,it is generally thought that the numerous neurotrophic factors can promote the growth and development in the nerve system.The insulin-like growth factor-1 is a peptide hormone,concerned closely with tissue differentiation、multiplication and maturity.IGF-1 could enhance the peripheral nerve's recovery by its non-elective neurotrophy effect.Search in votro,it is found that IGF-1 has the actions of neurotrophy and conservation on various kinds of central nerve cells effecting their growth、survivor and differentiation.Meanwhile,IGF-1 is proved able to inhibit the apoptosis caused by many stimulating factors such as ischemic、hypoxia、oxidative stress and so on.There are a few primary studies about the antioxidant function but few about the mechanism of protection to Schwann cell impaired from oxidation.
The investigation is aimed at probing into the antioxidant and protecting function of the insulin-like growth factor-1.The oxidization-damaged Schwann cells model induced by the peroxide hydrogen is used to investigate the mechanism of the IGF-1. Simultaneously,we offered the theoretical proofs for the insulin-like growth factor-1 to cure the neuropathy of peripheral nerve.
Method:In this subject,we chosen sciatic nerves and brachial nerves of neonatal Wistar rats younger than 1 week as experiment materials.They are cultured in liquid-DMEM/F12 containing 10%of the fetal calf serum(FCS)and 5%CO_2,37℃, saturated humidity.The Schwann cells cultured 72 hours long were divided into 3 experimental groups,meanwhile a blank group added.
1.Macro-observation:observe the Schwann cells of every group through the inverted phase contrast microscope after treated the cells for 12h.Comparing their appearance,quantities of survival cells and growth conditions with each groups,and record them.
2.Purify and identify Schwann cells:purity the Schwann cells by passage.After cultivating 3d we identified the cultured cells and calculated the purity of Schwann cells by the immunohistochemistry staining(S-100 antibody is used).
3.There were three cultured groups:one group was oxidization-damaged group treated with hydrogen whose final concentration was 100μmol/L,one was treated with peroxide hydrogen(100μmol/L H_2O_2)and the insulin-like growth factor-1(100ng/ml IGF-1),and another was control group only treated with culture liquid.
4.Investigating Schwann cells activity of different groups by MTT assay.
5.Measuring endocellular MDA and SOD by biochemistry assay.
6.Apoptosis within each group was detected by acridine orange staining.
7.Western blot was used to detect the expression of Bcl-2.
Through above experiments,we set up the oxidization-damaged Schwann cells model induced by hydrogen dioxide,and probed into the anti-oxidant protective action of the insulin-like growth factor-1 at the same time.
Result:Primary Schwann cells were round,suspending in the medium when inoculated at once.Most cells attached after 6 hours,and most cells began to turn into oval-shape.After 24 hours,the cells were dipolar with a power refraction of three-dimension and satiation and were shuttle-shape with two or three slender dendrites.After cultured 48-72 hours,there were polymerize that a lot of Schwann cells grew in group arranging end to end,side by side and palisade;the cells were assembled into pile called "cellular island".And immunohistochemistry(S-100) staining showed that the cells presented positive reaction.Another meager cell which was called fibroblast was bigger,irregular sight,and ancipital with some short ecphymas.Passage and purity the Schwann cells after 72h,and the purity increased obviously with 80%cells showed S-100 positive.12 hours after adding H_2O_2,we observed the change of the cellular shape under microscope:the dendrites withered, the cells swelled into roundness,vacuolization,the quantity of Schwann cells reduced, some cells took off the wall and floated in the culture liquid,the cells' three-dimensional scene was bad and refraction dropped,a large number of cell piece were obvious.But under the protection of the insulin-like growth factor-1,most cells were growing better with a powerful scene of three-dimension and the cells were scarcely withered and broken,but it was observed that the cells gathered obviously.In the control group,the cells' growing station was well.
The evaluation of different factor-treated groups was following:
1.Analyzing antioxidant function of the insulin-like growth factor-1(IGF-1)by MTT indicates:The IGF-1 can obviously resist the oxidization induced by the peroxide hydrogen.Compared with the hydrogen damaged group,there is a higher cellular livability in the IGF-1 treated group.There is statistical significance between them(P＜0.05),meanwhile the cellular livability in the IGF-1 treated group is lower than the normal group and there is also statistical significant(P＜0.05).
2.Biochemical measure indicates:Compared with the damaged group,the insulin-like growth factor-1 kept the endocellular superoxide dismutase(SOD)in high level.It was obvious that the insulin-like growth factor-1 resisted SOD reducing caused by the peroxide hydrogen though this level was lower than the control group. Both had statistics significance(P＜0.05).
3.Biochemical measure indicates:compared with the normal control group,the peroxide hydrogen caused lipid-hydrogen piled up inside the cells,that the MDA increased;but the insulin-like growth factor-1 obviously reduced this kind of pileup (P＜0.05).In the normal control group there was less endocellular lipid peroxide.
4.Viewed the cells stained by acridine orange through fluorescence microscope:the apoptosis cells turned round and smaller;apoptotic body appeared,yellow pykno-thickness collected in the cell nucleus and the cytoplasm showed jacinth.The normal group cells were fusiform shape with round or ellipse bright green nucleus and mignonette cytoplasm.The damaged group cells decreased and apoptosis obviously;but the protected group better.
5.Western blot expressed the level of Bcl-2 in each group:in the damaged group it decreased obviously,while it up-regulated after adding IGF-1(P＜0.05),the normal cells expressed the most.
Conclusion:We made the peroxide hydrogen-damaged Schwann cells model with the primary cultured Schwann cells.From the investigation we discovered that the insulin-like growth factor-1 resisted the oxidization damage induced by the peroxide hydrogen visibly.The peroxide hydrogen caused that the endocellular free radical increased,and the lipid activity of SOD reduced and the endocellular lipid-hydrogen increased.The increased free radicals resulted finally apoptosis through inhibiting the expression of Bcl-2 protein which resists to apoptosis.However, the IGF-1 can reduce the content of endocellular lipid hydrogen-MDA,maintain the higher level of SOD and up-regulation Bcl-2 to avoid the failure incident.
These results showed that the insulin-like growth factor-1's function protecting the Schwann cells by resisting the oxidization damage and the antioxidant molecular mechanism through the signal pathway of the oxidization damage.And it analyzed and confirmed the way in which the insulin-like growth factor-1 resisted the oxidization damage for the first time,provided the theoretical foundations for the neurotrophic factors such as the insulin-like growth factor-1 to cure the neuropathy of peripheral nerves.
目前,应用神经营养因子促进神经系统生长发育的研究已经得到人们广泛的认可。胰岛素样生长因子—1(insulin-like growth factor-1,IGF-1)是一种肽能神经生长因子,与组织分化、增殖和成熟密切相关。IGF-1具有非选择性神经营养作用,能促进外周神经再生。体外研究发现,IGF-1对各种中枢神经细胞均有神经营养和保护作用,能够影响神经细胞的生长、存活和分化。同时,大量实验证实,IGF-1对多种刺激因素(如缺血、缺氧,氧化应激等)引起的细胞凋亡有抑制作用。IGF-1的抗氧化作用已有初步研究,但对周围神经中Schwann细胞氧化损伤的保护作用的机理尚未见报道。
本课题通过建立过氧化氢诱导的Schwann细胞氧化损伤模型,并在此模型的基础上,观察了神经因子IGF-1对氧化损伤的Schwann细胞的保护作用,通过MTT、生化检测、吖叮橙染色、Western blot等方法进行细胞活力测定、酶活力分析、凋亡率的分析以及凋亡蛋白Bcl-2表达水平的测定,探讨IGF-1抗氧化损伤、抗凋亡的作用机理,为揭示IGF-1治疗周围神经病变的机制提供理论依据。
方法:本课题从新生一周内Wistar大鼠的坐骨神经和臂丛神经获取Schwann细胞,用含10%胎牛血清(FCS)的DMEM/F12培养液于5%CO_2、37℃、饱和湿度的细胞培养箱中培养。Schwann细胞培养72小时后,分为3个实验组,另设一空白对照组。
1.大体观察在加入H_2O_2损伤因素与IGF-1保护因素后12h时,倒置相差显微镜下观察Schwann细胞的大体形态、贴壁数量和生长状态等情况,并照相记录。
2.Schwann细胞的纯化及鉴定利用传代法纯化Schwann细胞。实验前Schwann细胞爬片作S-100免疫组化染色,鉴定Schwann细胞。
3.实验分组将培养细胞分为3组:①过氧化氢损伤组:在培养基中加入终浓度为100μmol/L的H_2O_2;②IGF-1保护组:100μmol/L的H_2O_2+100ng/ml的IGF-1;③正常组(不加任何因素);同时设立仅加培养液的空白对照组。
4.MTT法进行胰岛素样生长因子-1(IGF-1)抗过氧化氢氧化损伤的作用分析。
5.生化检测细胞内脂质过氧化物MDA和超氧化物歧化酶SOD的水平。
6.应用吖叮橙荧光染色观察、比较过氧化氢损伤组、IGF-1保护组和正常对照组细胞的存活、凋亡情况。
7.正常对照组、过氧化氢损伤组和IGF-1保护组细胞提取总蛋白,通过SDS-PAGE及蛋白印迹(Western blot)检测凋亡蛋白Bcl-2的表达水平变化情况。
通过以上的实验,建立了过氧化氢诱导Schwann细胞的氧化损伤模型,在此模型的基础上探讨IGF-1对Schwann细胞抗氧化损伤、抗凋亡的作用机理。
结果:刚接种的原代Schwann细胞呈圆形,悬浮在培养液中,接种6h后大部分细胞贴壁,部分细胞开始呈椭圆形,24h后变成双极,胞体饱满,立体感强,少数呈三角形,伸出三个突起。培养48～72h后,出现聚合现象,许多细胞聚合在一起,呈端对端、肩并肩、漩涡状或栅栏状排列,在细胞密度较低时,则聚集成堆,形成“细胞岛”,免疫细胞化学染色为S-100阳性;另一种细胞稍大,外形不规则,呈扁平状,突起短而多,核较浅,为成纤维细胞,所占比例较低。培养72小时后进行传代、纯化Schwann细胞,镜下观察Schwann细胞的纯度明显增加,免疫化学染色显示80%以上的细胞S-100呈阳性反应。加入H_2O_2 12小时后,在倒置显微镜下观察发现细胞的形态发生改变,细胞突起萎缩,胞体肿胀变圆、空泡化变,细胞数量减少,部分细胞脱壁,漂浮于培养液中,胞体立体感差,折光度下降,并可见大量细胞碎片;IGF-1保护组的细胞生长状态良好,突起伸展,细胞存活数量多,胞体较饱满、立体感较强,少见细胞皱缩破碎现象,但细胞聚团现象较明显;正常组的细胞胞体梭形、折光度好,突起较长,细胞数量明显较多,偶有漂浮死细胞。
不同实验组检测结果如下:
1.MTT法检测IGF-1抗氧化损伤的作用分析表明:IGF-1可明显拮抗过氧化氢诱导的氧化损伤,与过氧化氢损伤组相比,IGF-1保护组可维持较高的细胞存活率,二者之间有统计学意义(P＜0.05)。同时,IGF-1保护组的细胞存活率低于正常组,二者之间亦有统计学意义。
2.生化指标SOD检测结果表明:与过氧化氢损伤组相比,IGF-1保护组细胞内超氧化物歧化酶(SOD)维持在较高的活力水平,表明IGF-1可明显拮抗过氧化氢引起的SOD降低;同时低于正常对照组,有统计学意义(P＜0.05)。
3.生化指标MDA检测结果表明:与正常组相比,过氧化氢引起细胞内脂质过氧化物的大量堆积,即MDA含量增加;而IGF-1可明显降低细胞内脂质过氧化物的含量(P＜0.05)。
4.吖叮橙荧光染色法在荧光显微镜下观察到,发生凋亡的阳性细胞呈圆形,体积明显缩小,细胞核内可见致密浓染的黄色荧光和凋亡小体,胞质为火焰样橘红色荧光;正常组的Schwann细胞呈梭形,胞核圆形、椭圆形,呈亮绿色,胞质为灰绿色和橙红色,亦可见少量的凋亡前期细胞。损伤组细胞量明显减少,凋亡形态典型;保护组存活细胞量增多,细胞状态明显好转。
5.蛋白印迹Western blot显示抗凋亡蛋白Bcl-2的表达情况:过氧化氢损伤组细胞中抗凋亡蛋白Bcl-2的表达明显下调,而加入IGF-1保护后Bcl-2蛋白表达上调(P＜0.05),正常组的蛋白表达量最高。
结论:我们以原代培养的Schwann细胞为实验材料,利用过氧化氢诱导Schwann细胞建立氧化损伤模型,在此基础上,观察过氧化氢诱导Schwann细胞的氧化损伤作用及神经因子IGF-1对氧化损伤的Schwann细胞的保护作用。实验结果显示:过氧化氢损伤能够引起细胞内自由基的增多,SOD活力的降低,脂质过氧化物的增多,并且自由基的增多能通过信号通路上抑制凋亡蛋白Bcl-2的表达而最终引起细胞的凋亡;IGF-1不仅能降低细胞内脂质过氧化物(MDA)的含量,维持细胞内超氧化物歧化酶(SOD)的含量,还能通过上调抗凋亡蛋白Bcl-2的表达来抑制细胞死亡的发生,证明IGF-1可以明显拮抗过氧化氢对Schwann细胞的氧化损伤作用。
本研究探讨了IGF-1对Schwann细胞氧化损伤的保护作用,从氧化损伤信号转导通路方面探讨了IGF-1的抗氧化、抗凋亡作用的分子机制,为研究周围神经病变中自由基的分子作用机制以及IGF-1等神经营养因子在在周围神经病变中的治疗作用提供了一定的理论依据。
Purpose:Schwann cell is a major glial cell of peripheral nerve system on architecture and function.It plays an important part in the regeneration and functional restoration after the nerve injured.For many years,the pathological changes of peripheral nerve affect the people's living quality badly,which is a very important and complicate process in neural system diseases.Recently,researchers found that one of the pathogenesis is that oxidative irritation can make free radical increase,which cause the apoptosis of Schwann cells.Schwann cell can wrap the axon to form myelinated nerve fiber or nonmyelinated nerve fiber.It can secrete many kinds of neurotrophic factors which are concerned with development and regeneration of nerve.Recently, more and more people apply external assists:such as neurotrophic factor,Chinese traditional medicine,electromagnetism,silica gel and other grafts to enhance chwann cells' function,then to cure the peripheral nerve injuries.
Nowadays,it is generally thought that the numerous neurotrophic factors can promote the growth and development in the nerve system.The insulin-like growth factor-1 is a peptide hormone,concerned closely with tissue differentiation、multiplication and maturity.IGF-1 could enhance the peripheral nerve's recovery by its non-elective neurotrophy effect.Search in votro,it is found that IGF-1 has the actions of neurotrophy and conservation on various kinds of central nerve cells effecting their growth、survivor and differentiation.Meanwhile,IGF-1 is proved able to inhibit the apoptosis caused by many stimulating factors such as ischemic、hypoxia、oxidative stress and so on.There are a few primary studies about the antioxidant function but few about the mechanism of protection to Schwann cell impaired from oxidation.
The investigation is aimed at probing into the antioxidant and protecting function of the insulin-like growth factor-1.The oxidization-damaged Schwann cells model induced by the peroxide hydrogen is used to investigate the mechanism of the IGF-1. Simultaneously,we offered the theoretical proofs for the insulin-like growth factor-1 to cure the neuropathy of peripheral nerve.
Method:In this subject,we chosen sciatic nerves and brachial nerves of neonatal Wistar rats younger than 1 week as experiment materials.They are cultured in liquid-DMEM/F12 containing 10%of the fetal calf serum(FCS)and 5%CO_2,37℃, saturated humidity.The Schwann cells cultured 72 hours long were divided into 3 experimental groups,meanwhile a blank group added.
1.Macro-observation:observe the Schwann cells of every group through the inverted phase contrast microscope after treated the cells for 12h.Comparing their appearance,quantities of survival cells and growth conditions with each groups,and record them.
2.Purify and identify Schwann cells:purity the Schwann cells by passage.After cultivating 3d we identified the cultured cells and calculated the purity of Schwann cells by the immunohistochemistry staining(S-100 antibody is used).
3.There were three cultured groups:one group was oxidization-damaged group treated with hydrogen whose final concentration was 100μmol/L,one was treated with peroxide hydrogen(100μmol/L H_2O_2)and the insulin-like growth factor-1(100ng/ml IGF-1),and another was control group only treated with culture liquid.
4.Investigating Schwann cells activity of different groups by MTT assay.
5.Measuring endocellular MDA and SOD by biochemistry assay.
6.Apoptosis within each group was detected by acridine orange staining.
7.Western blot was used to detect the expression of Bcl-2.
Through above experiments,we set up the oxidization-damaged Schwann cells model induced by hydrogen dioxide,and probed into the anti-oxidant protective action of the insulin-like growth factor-1 at the same time.
Result:Primary Schwann cells were round,suspending in the medium when inoculated at once.Most cells attached after 6 hours,and most cells began to turn into oval-shape.After 24 hours,the cells were dipolar with a power refraction of three-dimension and satiation and were shuttle-shape with two or three slender dendrites.After cultured 48-72 hours,there were polymerize that a lot of Schwann cells grew in group arranging end to end,side by side and palisade;the cells were assembled into pile called "cellular island".And immunohistochemistry(S-100) staining showed that the cells presented positive reaction.Another meager cell which was called fibroblast was bigger,irregular sight,and ancipital with some short ecphymas.Passage and purity the Schwann cells after 72h,and the purity increased obviously with 80%cells showed S-100 positive.12 hours after adding H_2O_2,we observed the change of the cellular shape under microscope:the dendrites withered, the cells swelled into roundness,vacuolization,the quantity of Schwann cells reduced, some cells took off the wall and floated in the culture liquid,the cells' three-dimensional scene was bad and refraction dropped,a large number of cell piece were obvious.But under the protection of the insulin-like growth factor-1,most cells were growing better with a powerful scene of three-dimension and the cells were scarcely withered and broken,but it was observed that the cells gathered obviously.In the control group,the cells' growing station was well.
The evaluation of different factor-treated groups was following:
1.Analyzing antioxidant function of the insulin-like growth factor-1(IGF-1)by MTT indicates:The IGF-1 can obviously resist the oxidization induced by the peroxide hydrogen.Compared with the hydrogen damaged group,there is a higher cellular livability in the IGF-1 treated group.There is statistical significance between them(P＜0.05),meanwhile the cellular livability in the IGF-1 treated group is lower than the normal group and there is also statistical significant(P＜0.05).
2.Biochemical measure indicates:Compared with the damaged group,the insulin-like growth factor-1 kept the endocellular superoxide dismutase(SOD)in high level.It was obvious that the insulin-like growth factor-1 resisted SOD reducing caused by the peroxide hydrogen though this level was lower than the control group. Both had statistics significance(P＜0.05).
3.Biochemical measure indicates:compared with the normal control group,the peroxide hydrogen caused lipid-hydrogen piled up inside the cells,that the MDA increased;but the insulin-like growth factor-1 obviously reduced this kind of pileup (P＜0.05).In the normal control group there was less endocellular lipid peroxide.
4.Viewed the cells stained by acridine orange through fluorescence microscope:the apoptosis cells turned round and smaller;apoptotic body appeared,yellow pykno-thickness collected in the cell nucleus and the cytoplasm showed jacinth.The normal group cells were fusiform shape with round or ellipse bright green nucleus and mignonette cytoplasm.The damaged group cells decreased and apoptosis obviously;but the protected group better.
5.Western blot expressed the level of Bcl-2 in each group:in the damaged group it decreased obviously,while it up-regulated after adding IGF-1(P＜0.05),the normal cells expressed the most.
Conclusion:We made the peroxide hydrogen-damaged Schwann cells model with the primary cultured Schwann cells.From the investigation we discovered that the insulin-like growth factor-1 resisted the oxidization damage induced by the peroxide hydrogen visibly.The peroxide hydrogen caused that the endocellular free radical increased,and the lipid activity of SOD reduced and the endocellular lipid-hydrogen increased.The increased free radicals resulted finally apoptosis through inhibiting the expression of Bcl-2 protein which resists to apoptosis.However, the IGF-1 can reduce the content of endocellular lipid hydrogen-MDA,maintain the higher level of SOD and up-regulation Bcl-2 to avoid the failure incident.
These results showed that the insulin-like growth factor-1's function protecting the Schwann cells by resisting the oxidization damage and the antioxidant molecular mechanism through the signal pathway of the oxidization damage.And it analyzed and confirmed the way in which the insulin-like growth factor-1 resisted the oxidization damage for the first time,provided the theoretical foundations for the neurotrophic factors such as the insulin-like growth factor-1 to cure the neuropathy of peripheral nerves.
引文
1.Meyer G,KimB,van Golen C,et al.Cofilin activity during insulin-like growth factor-1 stimulated neuroblastoma cell motility[J].Cell Mol Life Sci,2005,62(4):461-470.
2.Raushan T.Kurmasheva,Peter J.Houghton.IGF-1 mediated survival pathways in normal and malignant cells[J].Biochim Biophys Acta,2006,1766(1):1-22.
3.Senoo E,Tamaki N,Fujimoto E,et al.Effects of prelesioned peripheral nerve graft on nerve regeneration in the rat spinal cord[J].Neurosurgey,1998,42(6):1347-1356.
4.Ard MD,Bunge RP,Bunge MB.Comparison of the Schwann cell surface and Schwann cell extracellular matrix as promoters of neurite growth[J].Neurocytol,1987,16(4):539-555.
5.Carey DJ,Bunge RP.Factors influencing the release of proteins by cultured Schwann cells[J].Cell Biol,1981,91(3 Pt 1):666-672.
6.秦新月,董为伟.胰岛素样生长因子对少枝胶质细胞生长及髓鞘再生的影响[J].卒中与神经疾病,2002,9(5):259-262.
7.Hwang IK,Yoo KY,Park SK.,et al.Expression and changes of endogenous insulin-like growth factor-1 in neurons and glia in the gerbil hippocampus and dentate gyrus after ischemic insult[J].Neurochem Int,2004,45(1):149-156.
8.Naira Baregamian,M.D.,Jun Song,M.D.,Marc G.Jeschke,M.D.,et al.IGF-1 protects intestinal epithelial cells from oxidative stress-induced apoptosisl[J].Journal of Surgical Research,2006,136(1):31-37.
9.杨洁、高飞.活性氧与细胞凋亡的研究进展[J],国外医学肿瘤学分册.2002,29(4):248-251.
10.Low PA,Nickander KK.Oxygen free radical effects in sciatic nerve in experimental diabetes[J].Diabetes,1991,40(7):873-877.
11.Tang XQ,Feng JQ,Chen J.Et al.Protection of oxidative preconditioning against apoptosis induced by H_2O_2 in PC12 cells:mechanisms via MMP,ROS,and Bcl-2[J].Brain Res.2005,1057(1-2):57-64.
12.Hockenbery D.Defining apoptosis[J].Am J Pathol,1995,146(1):16-19.
13.姜泊.细胞凋亡基础与临床[M].人民军医出版社,1999,7(2):104-106.
14.von Harsdorf R,Li PF,Dietz R.Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis[J].Circulation,1999,99(22):2934-2941.
15.Wei YH,Lee HC.Oxidative stress mitochondrial DNA mutation and impairment of antioxidant enzymes in aging[J].Exp Biol Med (Maywood),2002,227(9):671-682.
16.Miller DJ,MacFartane NG.Intracelluar effects of free radicals and reactive oxygen soecies in cardiac muscle[J].J Hump Hypertens,1995,9(6):465-473.
17.Korsmeyer SJ,Shutter JR,Veis DJ,et al.Bcl-2/Bax:a rheostat that regulates an anti-oxidant pathway and cell death[J].Semin Cancer Biol,1993,4(6):327-332.
18.Aoshima H,Satoh T,Sakai N,et al.Generation of free radical during lipid hydroperoxide-triggered apoptosis in PC12h cells[J].Biochim Biophys Acta,1997,1345(1):35-42.
19.Chert QM,Tu VC,Wu Y,et al.Hydrogen peroxide dose dependent induction of cell death or hypertrophy in cardiomyocyte[J].ArchBiochemBiophys,2000,373(1):242-248.
20.钱淑凤,高桂生,郑翠玲.细胞凋亡与疾病[J].中国畜牧兽医,2007,34(9):88-91
21.Danial NN,Korsmeyer SJ.Cell death:critical control points[J].Cell,2004,116(2):205-219).
22.Peter ME,Krammer PH.The CD95(APO-1/Fas)DISC and beyond[J].Cell Death Differ.2003,10(1):26-35.
23.Micheau O,Tschopp J.Induction of TNF receptor Ⅰ-mediated apoptosis via two sequential signaling complexes[J].Cell.2003,114(2):181-190.
24.Hengartner MO.The biochemistry of apoptosis[J].Nature 2000,407(6805):770-776.
25.Leist M,Jaatrela M.Four deaths and a funeral:from caspases to alternative mechanisms[J].Nat Rev Mol Cell Biol,2001,2(8):589-598.
26.Jaattela M,Wschopp J.Caspase-independent cell death in T lymphocytes[J].Nat Immunol.2003,4(5):416-423.
27.Lemasters JJ,Nieminen AL,Qian T,et al.The mitochondrial permeability transition in cell death:a common mechanism in necrosis,apoptosis and autophage[J].Biocheim Biophys Acta,1998,1366(12):177-196.
28.程何祥,张荣庆,马颖艳,等.过氧化氢诱导乳鼠心肌细胞凋亡及半胱天冬酶-3抑制剂的作用[J].第四军医大学学报,2001,22(21):1953-1956.
29.Ashkenazi A,Dixit VM.Death receptors:signaling and modulation[J].Science,1998,281(5381):1305-1308.
30.吴小梅,陈宏山,金淑仪,等.银杏内酯抗过氧化氢致神经细胞损伤和对即早基因表达的影响[J].中国临床药理学与治疗学,2003,8(4):401-404.
31.李源,郑延松,臧益民,等.褪黑素对氧化损伤培养心肌细胞的保护作用[J].第四军医大学学报,2002,23(22):2060-2062.
32.Keller JN,Mattson MP.Roles of lipid peroxidatidation in modulation of cellular signaling pathways,cell dysfunction,an death in the nervous system[J].RevUeurosci,1998,9(2):105-116.
33.Vita JA,Treasure CB,Yeung AC,et al.Patients with evidence of coronary endothelial dysfunction as assessed by acetylcholine infusion demonstrate marked increase insensitivity to constrictor effects of catecholamines[J].Circulation,1992,85(4):1390-1397.
34.Li Y,Maher P,Schubert D.A role for 12-1ipoxygenase in nerve cell death caused by glutathione depletion[J].Neuron,1997,19(2):453-463.
35.韩彩萍,胡长林.胰岛素样生长因子-1的神经保护作用[J].国外医学脑血管疾病分册,2005,13(1):46-49.
36.Enari M,Sakahira H,Yokoyama H,et al.A caspase-activated DNase that degrades DNA during apoptosis and its inhibitor ICAD[J].Nature,1998,391(6662):43-50.
37.Samali A,Nordgren H,Orrenius S,et al.A comparative study of apoptosis and necrosis in HepG2 cells:oxidant induced caspase inactivation leads to necrosis[J].Biochem Biophys Res Commun,1999,255(1):6-11.
38.Senoo E,Tamaki N,Fujimoto E,et al.Effents of perlesioned peripheral nerve graft on nerve regenation in the rat sina cord[J].Neurosurgey,1998,42(6):1347-1356.
39.Ard MD,Bunque RP,et al.Comparision of the schwann cell surface and Schwann cell extracellular matrix as promoters of neurite growth [J].J Neurocytol,1987,16(4):539-555.
40.Carey DJ,Bunge RP.Factors influencing the release of proteins by cultured Schwann cells[J].J Cell Biol.1981,91(3Pt):666-672.
41.Purves T,Middlemas A,Agthong S,et al.A role for mitogen-activated protein kinases in the etiology of diabetic neuropathy[J].FASEB J,2001,15(13):2508-2514.
42.Cui Q,Harvey AR.At least two mechanisms are involved in the death of retinal ganglion cells following target ablation in neonatal rats[J].J Neurosci,1995,15(12):8143-8155.
43.Kwon D,Choi C,Lee J,et al.Hydrogen peroxide triggers the expression of Fas/Fasl in astrocytoma cell lines and augments apoptosis[J].J Neuroimmunol,2001,113(1):1-9.
44.Block ER.Hydrogen peroxide alters the physical state and function of the plasma membrane of pulmonary artery endothelial cells[J]. J Cell Physiol, 1991,146(3): 362-369.
45. Clemmons DR, Moses AC, Mckay MJ , et al. The combination of insulin-like growth factor 1 and insulin-like growth factor-binding protein-3 reduces insulin requirements in insulin-dependent type 1 diabetes: evidence for in vivo biological act ivity[J]. J Clin Endocrinol Metab, 2000, 85(4): 1518-1524.
46. Mathonnet M , Comte I, Lalloue F, et al. Insulin-like growth factor 1 induced survival of axotomized olfactory neurons in the chick[J]. Neurosci Lett, 2001, 308 (2) : 66-70.
47. Hwang IK, Yoo KY, Park SK. , et al. Expression and changes of endogenous insulin-like growth factor-1 in neurons and glia in the gerbil hippocampus and dentate gyrus after ischemic insult [J]. Neurochem Int, 2004, 45(1): 149 - 156.
48. Naira Baregamian, Jun Song, Marc G. Jeschke, et al. IGF—1 protects intestinal epithelial cells from oxidative stress-induced apoptosis[J]. Journal of Surgical Research 2006, 136(1):31 - 37.
49. Mosmann T. Rapid colorimeletic assay for cellular growth and Survival application to proliferation and cytoloxicity assays[J]. Immunol Methods, 1983, 65 (1-2): 55-63.
50.Chan PH. Role of oxidants in ischemia brain damage [J]. Stroke, 1996, 27 (6): 1124-1129.
51. Mugge A, Elwell J, Peterson TE, et al. Release of intact endothelium-derived relaxing factor depends on endothelial superoxide dismutase activity[J]. Am J Physiol Cell Physiol, 1991, 260(2Ptl):219-225.
52. Shackelford RE, Kaufmann WK, Paules RS. Oxidative stress and cell cycle checkpoint function[J]. Free Radic Biol Med, 2000, 28 (9): 1387-1404.
53.Kugiyama K,Kems SA,Morrisett JD,et al.Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins[J].Nature,1990,344(6262):160-162.
54.Kinnula VL,Everitt JI,Whorton AR,et al.Hydrogen peroxide production by alverlar typeⅡcells,alveolar macrophages and endothelial cells [J].Am J Physiol.1999,261(2):84-91.
55.Rashmi R,Kumar S,Karunagaran D.Human colon cancer cells lacking Bax resist curcumin-induced apoptosis and Bax requirement is dispensable with ectopic expression of Smac or downregulation of Bcl-XL[J].Carcinogenesis,2005,26(4):713-723.
56.Baker N L,Carlo Russo V,Bernard O,et al.Interactions between bcl-2and the IGF system control apoptosis in the developing mouse brain[J].Brain Res Dev Brain Res,1999,118(1-2):109-118.
57.Offen D,Shtaif B,Hadad D,et al.Protective effect of insulin-like growth factor-1 against dopamine-induced neurotoxicity in human and rodent neuronal cultures:possible imp lications for Parkinson's disease[J].Neurosci Lett,2001,316(3):129-132.
58.苏剑东,吴灵飞.NF-κB与细胞凋亡[J].世界华人消化杂志,2007,15(12):1411-1416.
59.Gleichmann M,Weller M,Schulz J B.Insulin-like growth factor-1-mediated protection from neuronal apoptosis is linked to phosphorylation of the pro-apop totic protein BAD but not to inhibition of cytochrome c translocation in rat cerebellar neurons[J].Neurosci Lett,2000,282(1-2):69-72.
60.Cao Y,Gunn A J,Bennet L,et al.lnsulin-like growth factor(IGF)-1suppresses oligodendrocyte caspase-3 activation and increases glial proliferation after ischemia in near-tirm fetal sheep[J].Cereb Blood Flow Metab,2003,23(6):739-747.
2.Raushan T.Kurmasheva,Peter J.Houghton.IGF-1 mediated survival pathways in normal and malignant cells[J].Biochim Biophys Acta,2006,1766(1):1-22.
3.Senoo E,Tamaki N,Fujimoto E,et al.Effects of prelesioned peripheral nerve graft on nerve regeneration in the rat spinal cord[J].Neurosurgey,1998,42(6):1347-1356.
4.Ard MD,Bunge RP,Bunge MB.Comparison of the Schwann cell surface and Schwann cell extracellular matrix as promoters of neurite growth[J].Neurocytol,1987,16(4):539-555.
5.Carey DJ,Bunge RP.Factors influencing the release of proteins by cultured Schwann cells[J].Cell Biol,1981,91(3 Pt 1):666-672.
6.秦新月,董为伟.胰岛素样生长因子对少枝胶质细胞生长及髓鞘再生的影响[J].卒中与神经疾病,2002,9(5):259-262.
7.Hwang IK,Yoo KY,Park SK.,et al.Expression and changes of endogenous insulin-like growth factor-1 in neurons and glia in the gerbil hippocampus and dentate gyrus after ischemic insult[J].Neurochem Int,2004,45(1):149-156.
8.Naira Baregamian,M.D.,Jun Song,M.D.,Marc G.Jeschke,M.D.,et al.IGF-1 protects intestinal epithelial cells from oxidative stress-induced apoptosisl[J].Journal of Surgical Research,2006,136(1):31-37.
9.杨洁、高飞.活性氧与细胞凋亡的研究进展[J],国外医学肿瘤学分册.2002,29(4):248-251.
10.Low PA,Nickander KK.Oxygen free radical effects in sciatic nerve in experimental diabetes[J].Diabetes,1991,40(7):873-877.
11.Tang XQ,Feng JQ,Chen J.Et al.Protection of oxidative preconditioning against apoptosis induced by H_2O_2 in PC12 cells:mechanisms via MMP,ROS,and Bcl-2[J].Brain Res.2005,1057(1-2):57-64.
12.Hockenbery D.Defining apoptosis[J].Am J Pathol,1995,146(1):16-19.
13.姜泊.细胞凋亡基础与临床[M].人民军医出版社,1999,7(2):104-106.
14.von Harsdorf R,Li PF,Dietz R.Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis[J].Circulation,1999,99(22):2934-2941.
15.Wei YH,Lee HC.Oxidative stress mitochondrial DNA mutation and impairment of antioxidant enzymes in aging[J].Exp Biol Med (Maywood),2002,227(9):671-682.
16.Miller DJ,MacFartane NG.Intracelluar effects of free radicals and reactive oxygen soecies in cardiac muscle[J].J Hump Hypertens,1995,9(6):465-473.
17.Korsmeyer SJ,Shutter JR,Veis DJ,et al.Bcl-2/Bax:a rheostat that regulates an anti-oxidant pathway and cell death[J].Semin Cancer Biol,1993,4(6):327-332.
18.Aoshima H,Satoh T,Sakai N,et al.Generation of free radical during lipid hydroperoxide-triggered apoptosis in PC12h cells[J].Biochim Biophys Acta,1997,1345(1):35-42.
19.Chert QM,Tu VC,Wu Y,et al.Hydrogen peroxide dose dependent induction of cell death or hypertrophy in cardiomyocyte[J].ArchBiochemBiophys,2000,373(1):242-248.
20.钱淑凤,高桂生,郑翠玲.细胞凋亡与疾病[J].中国畜牧兽医,2007,34(9):88-91
21.Danial NN,Korsmeyer SJ.Cell death:critical control points[J].Cell,2004,116(2):205-219).
22.Peter ME,Krammer PH.The CD95(APO-1/Fas)DISC and beyond[J].Cell Death Differ.2003,10(1):26-35.
23.Micheau O,Tschopp J.Induction of TNF receptor Ⅰ-mediated apoptosis via two sequential signaling complexes[J].Cell.2003,114(2):181-190.
24.Hengartner MO.The biochemistry of apoptosis[J].Nature 2000,407(6805):770-776.
25.Leist M,Jaatrela M.Four deaths and a funeral:from caspases to alternative mechanisms[J].Nat Rev Mol Cell Biol,2001,2(8):589-598.
26.Jaattela M,Wschopp J.Caspase-independent cell death in T lymphocytes[J].Nat Immunol.2003,4(5):416-423.
27.Lemasters JJ,Nieminen AL,Qian T,et al.The mitochondrial permeability transition in cell death:a common mechanism in necrosis,apoptosis and autophage[J].Biocheim Biophys Acta,1998,1366(12):177-196.
28.程何祥,张荣庆,马颖艳,等.过氧化氢诱导乳鼠心肌细胞凋亡及半胱天冬酶-3抑制剂的作用[J].第四军医大学学报,2001,22(21):1953-1956.
29.Ashkenazi A,Dixit VM.Death receptors:signaling and modulation[J].Science,1998,281(5381):1305-1308.
30.吴小梅,陈宏山,金淑仪,等.银杏内酯抗过氧化氢致神经细胞损伤和对即早基因表达的影响[J].中国临床药理学与治疗学,2003,8(4):401-404.
31.李源,郑延松,臧益民,等.褪黑素对氧化损伤培养心肌细胞的保护作用[J].第四军医大学学报,2002,23(22):2060-2062.
32.Keller JN,Mattson MP.Roles of lipid peroxidatidation in modulation of cellular signaling pathways,cell dysfunction,an death in the nervous system[J].RevUeurosci,1998,9(2):105-116.
33.Vita JA,Treasure CB,Yeung AC,et al.Patients with evidence of coronary endothelial dysfunction as assessed by acetylcholine infusion demonstrate marked increase insensitivity to constrictor effects of catecholamines[J].Circulation,1992,85(4):1390-1397.
34.Li Y,Maher P,Schubert D.A role for 12-1ipoxygenase in nerve cell death caused by glutathione depletion[J].Neuron,1997,19(2):453-463.
35.韩彩萍,胡长林.胰岛素样生长因子-1的神经保护作用[J].国外医学脑血管疾病分册,2005,13(1):46-49.
36.Enari M,Sakahira H,Yokoyama H,et al.A caspase-activated DNase that degrades DNA during apoptosis and its inhibitor ICAD[J].Nature,1998,391(6662):43-50.
37.Samali A,Nordgren H,Orrenius S,et al.A comparative study of apoptosis and necrosis in HepG2 cells:oxidant induced caspase inactivation leads to necrosis[J].Biochem Biophys Res Commun,1999,255(1):6-11.
38.Senoo E,Tamaki N,Fujimoto E,et al.Effents of perlesioned peripheral nerve graft on nerve regenation in the rat sina cord[J].Neurosurgey,1998,42(6):1347-1356.
39.Ard MD,Bunque RP,et al.Comparision of the schwann cell surface and Schwann cell extracellular matrix as promoters of neurite growth [J].J Neurocytol,1987,16(4):539-555.
40.Carey DJ,Bunge RP.Factors influencing the release of proteins by cultured Schwann cells[J].J Cell Biol.1981,91(3Pt):666-672.
41.Purves T,Middlemas A,Agthong S,et al.A role for mitogen-activated protein kinases in the etiology of diabetic neuropathy[J].FASEB J,2001,15(13):2508-2514.
42.Cui Q,Harvey AR.At least two mechanisms are involved in the death of retinal ganglion cells following target ablation in neonatal rats[J].J Neurosci,1995,15(12):8143-8155.
43.Kwon D,Choi C,Lee J,et al.Hydrogen peroxide triggers the expression of Fas/Fasl in astrocytoma cell lines and augments apoptosis[J].J Neuroimmunol,2001,113(1):1-9.
44.Block ER.Hydrogen peroxide alters the physical state and function of the plasma membrane of pulmonary artery endothelial cells[J]. J Cell Physiol, 1991,146(3): 362-369.
45. Clemmons DR, Moses AC, Mckay MJ , et al. The combination of insulin-like growth factor 1 and insulin-like growth factor-binding protein-3 reduces insulin requirements in insulin-dependent type 1 diabetes: evidence for in vivo biological act ivity[J]. J Clin Endocrinol Metab, 2000, 85(4): 1518-1524.
46. Mathonnet M , Comte I, Lalloue F, et al. Insulin-like growth factor 1 induced survival of axotomized olfactory neurons in the chick[J]. Neurosci Lett, 2001, 308 (2) : 66-70.
47. Hwang IK, Yoo KY, Park SK. , et al. Expression and changes of endogenous insulin-like growth factor-1 in neurons and glia in the gerbil hippocampus and dentate gyrus after ischemic insult [J]. Neurochem Int, 2004, 45(1): 149 - 156.
48. Naira Baregamian, Jun Song, Marc G. Jeschke, et al. IGF—1 protects intestinal epithelial cells from oxidative stress-induced apoptosis[J]. Journal of Surgical Research 2006, 136(1):31 - 37.
49. Mosmann T. Rapid colorimeletic assay for cellular growth and Survival application to proliferation and cytoloxicity assays[J]. Immunol Methods, 1983, 65 (1-2): 55-63.
50.Chan PH. Role of oxidants in ischemia brain damage [J]. Stroke, 1996, 27 (6): 1124-1129.
51. Mugge A, Elwell J, Peterson TE, et al. Release of intact endothelium-derived relaxing factor depends on endothelial superoxide dismutase activity[J]. Am J Physiol Cell Physiol, 1991, 260(2Ptl):219-225.
52. Shackelford RE, Kaufmann WK, Paules RS. Oxidative stress and cell cycle checkpoint function[J]. Free Radic Biol Med, 2000, 28 (9): 1387-1404.
53.Kugiyama K,Kems SA,Morrisett JD,et al.Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins[J].Nature,1990,344(6262):160-162.
54.Kinnula VL,Everitt JI,Whorton AR,et al.Hydrogen peroxide production by alverlar typeⅡcells,alveolar macrophages and endothelial cells [J].Am J Physiol.1999,261(2):84-91.
55.Rashmi R,Kumar S,Karunagaran D.Human colon cancer cells lacking Bax resist curcumin-induced apoptosis and Bax requirement is dispensable with ectopic expression of Smac or downregulation of Bcl-XL[J].Carcinogenesis,2005,26(4):713-723.
56.Baker N L,Carlo Russo V,Bernard O,et al.Interactions between bcl-2and the IGF system control apoptosis in the developing mouse brain[J].Brain Res Dev Brain Res,1999,118(1-2):109-118.
57.Offen D,Shtaif B,Hadad D,et al.Protective effect of insulin-like growth factor-1 against dopamine-induced neurotoxicity in human and rodent neuronal cultures:possible imp lications for Parkinson's disease[J].Neurosci Lett,2001,316(3):129-132.
58.苏剑东,吴灵飞.NF-κB与细胞凋亡[J].世界华人消化杂志,2007,15(12):1411-1416.
59.Gleichmann M,Weller M,Schulz J B.Insulin-like growth factor-1-mediated protection from neuronal apoptosis is linked to phosphorylation of the pro-apop totic protein BAD but not to inhibition of cytochrome c translocation in rat cerebellar neurons[J].Neurosci Lett,2000,282(1-2):69-72.
60.Cao Y,Gunn A J,Bennet L,et al.lnsulin-like growth factor(IGF)-1suppresses oligodendrocyte caspase-3 activation and increases glial proliferation after ischemia in near-tirm fetal sheep[J].Cereb Blood Flow Metab,2003,23(6):739-747.